1. Field of the Invention
The invention relates to a method for producing open-chain sophorolipids by fermentation with a yeast.
2. Description of the Prior Art
Biosurfactants are microbially produced, renewable, surface-active compounds that are increasingly being used for oil and mineral recovery, and in detergents, cosmetics, and anti-microbial formulations (Georgiou et al. Nature Biotechnol. 1992. 10:60-65; Cooper and Zajic. Adv. Appl. Microbiol. 1980. 26:229-253; Dembitsky Lipids. 2004. 39:933-953; and Banat et al. R. Appl. Microbiol. Biotechnol. 2010. 87:427-444). The annual worldwide production of surfactants is currently about 10 million tons, and are mostly derived from petroleum feedstocks, but production from renewable sources is now of great interest Banat et al., ibid). Included in the glycolipid-type biosurfactants are sophorolipids from various Starmerella yeast species (Van Bogaert et al. 2011. Process Biochem. 46:821-833; Rau et al. 2001. Ind. Crops Prod. 13:85-92; Develter and Lauryssen. 2010. Eur. J. Lipid Sci. Technol. 112:628-638; and Baccile et al. 2010. Green Chem. 12:1564-1567), which consist of the disaccharide sophorose linked to a long chain hydroxy fatty acid. Among yeasts of the Starmerella clade (genetically related group of species) that have been examined, the greatest yield of sophorolipids has been reported from Candida apicola and Starmerella bombicola (Van Bogaert et al. 2007 Appl. Microbiol. Biotechnol. 76:23-34). These sophorolipids are a partially acetylated 2-O-β-D-glucopyranosyl-D-glucopyranose unit attached β-glycosidically to 17-L-hydroxyoctadecanoic or 17-L-hydroxy-Δ9-octadecenoic acid (Tulloch et al. 1962. Can. J. Chem. 40:1326-1338; and Tulloch et al. 1968. Can. J. Chem. 46:345-348) and can be acetylated on the 6′- and/or 6″-positions. The hydroxy fatty acid is generally 16 or 18 carbon atoms, and may contain one or more unsaturated bonds (Asmer et al. 1988. J. Am. Oil Chem. Soc. 65:1460-1466; and Davila et al. 1993. J. Chromatogr. 648:139-149). The fatty acid carboxyl group is either free (acidic or open form) or internally esterified at the 4″-position (lactone form). The 1,4″-lactone form of the sophorolipids are nonionic surfactants with a critical micelle concentration (CMC) of 40-100 μg·mL−1 and are reported to lower the surface tension of water by 30-40 mN·m−1, making them especially useful as emulsifiers for oil/water mixtures (Van Bogaert et al. 2007. ibid).
Interest in sophorolipids is not limited to production of surfactants. The unique chemical structure of sophorolipids can serve as the basis for synthesizing certain hydroxy fatty acids and other compounds (Van Bogaert et al. 2007. ibid). Perhaps of greater interest are reports that these glycolipids have antimicrobial activity against certain yeasts (Ito et al. 1980. Agric Biol Chem. 44:2221-2223), plant pathogenic fungi (Yoo et al. 2005. J Microbiol Biotechnol. 15:1164-1169) and bacteria (Mager et al. 1987. European Patent no. 0209783; and Lang et al. 1989. Fett Wiss Technol Fat Sci Technol. 91:363-366). Furthermore, Shah et al. (2005. Antimicrob Agents Chemother. 49:4093-4100) showed inhibition of the HIV virus by sophorolipids, and Chen et al. (2006. Enzyme Microb Technol. 39:501-506) provided evidence that the compounds have anti-cancer activity.
As noted above, sophorolipids are synthesized by a phylogenetically diverse group of yeasts. The earliest report appears to be that of Gorin et al. (1961. Can J Chem. 39:846-855), who demonstrated sophorolipid biosynthesis by the anamorphic ascomycetous yeast Candida apicola, which was initially identified as C. magnoliae. Later, Spencer et al. (1970. Antonie van Leeuwenhoek. 36:129-133) showed sophorolipid production by C. bombicola, and Konoshi et al. (2008. J Oleo Sci. 57:359-369) reported C. batistae to also form sophorolipids. The preceding three Candida species are closely related, but sophorolipid biosynthesis was also demonstrated for the less closely related Wickerhamiella domercqiae (Chen et al., 2006. ibid) as well as for the basidiomycetous yeast Rhodotorula bogoriensis (Tulloch et al. 1968. ibid).
Phylogenetic analysis of sequences for the D1/D2 domains of the nuclear large subunit ribosomal RNA gene has shown that C. apicola and C. bombicola are members of a clade that is well separated from other ascomycetous yeasts (Kurtzman & Robnett. 1998. Antonie van Leeuwenhoek. 73:331-371). Candida bombicola is the first member of the clade for which ascospore formation was discovered and the species was reassigned to the teleomorphic genus Starmerella (Rosa & Lachance. 1998. Int J Syst Bacteriol. 48:1413-1417). With the application of sequence analysis to yeast identification, the group of yeasts related to S. bombicola, now termed the Starmerella clade, has increased markedly in the past decade to over 40 species with many not yet described and presently recognized only from their gene sequences, which have been deposited in GenBank. Candida apicola, C. batistae and S. bombicola are the only members of the Starmerella clade that have been reported to produce sophorolipids.
The S. bombicola sophorolipids are nearly identical to those of C. apicola (Spencer et al. 1970. ibid). Chen et al. (2006. ibid) observed more than six sophorolipids from Wickerhamiella domericqiae, and identified one as 17-L-(-oxy)-octadecanoic acid 1,4″-lactone 6′,6″-diacetate, that is also the major sophorolipid from C. apicola and C. bombicola. Tulloch et al. (1968. ibid) also discovered similar sophorolipids from C. bogoriensis, but that differ in the hydroxy fatty acid moiety, which in this instance is 13-hydroxydocosanoic acid (J. Biol Chem. 1979. 254:1944-1950).
However, despite these and other advances, the need remains for improved sophorolipids and methods for their production.